Method for analyzing unicellular inclusion

Abstract

The invention relates to a method for analyzing a unicellular inclusion. The method comprises main steps of 1, by means of a portable single-particle pipettor, preparing a 96 or 384 orifice plate which loads a single coding microball; 2, sorting a unicell by means of a fluorescent activation flow sorting instrument, or separating a rare cell to a micro-orifice plate by means of the pipettor, thereby realizing quick one-to-one pairing between a unicell and a single microball; and 3, converting the inclusion information in the unicell to DNA sequence information by means of the coding microball,and analyzing the sequencing data by means of high-flux sequencing technology and bioinformatics. The method of the invention can realize high-efficiency stable low-cost separation of the single coding microball and has advantages of low technical threshold, effective overcoming of Poisson distribution, wide target range, controllable flux, low cost, etc. Furthermore the developed single-particlepipettor can be used for capturing and sequencing of the rare cell and can be widely used for the fields such as basic research and clinical diagnosis unicell analysis.

Description

technical field [0001] The invention belongs to the field of single-cell sequencing, in particular to a method for analyzing single-cell content. Background technique [0002] The heterogeneity of cells is a ubiquitous life phenomenon. As an independent living entity, the properties and differences displayed by a single cell play a vital role in the development of the entire living system. Every tissue and organ in the human body contains a variety of cell types, and each type of cell will change with the life activity of the organism. If thousands of single cells are studied, it will be blurred Therefore, it is extremely important to understand the working principles of complex organisms, the life functions and immune responses of each cell type to reveal the working mechanism of human tissues and organs and the laws of gene regulation. For example, malignant tumors that cause human cancer are highly heterogeneous tissues, composed of tumor cells of various phenotypes, and...

AI Technical Summary

Problems solved by technology

[0005] The second technical problem is the amplification of micro-contents in single cells, and how to label each cell during sample preparation in order to achieve high-throughput single-cell sequencing, that is, to introduce cell codes; There are deviations in the process. How to label the information of each transcript in a single cell, that is, to introduce molecular codes, and how to integrate cellular codes and molecular codes to achieve accurate quantification of cell contents have become the methodologies of scientific researchers in recent years. focus on innovation

[0006] The third technical challenge is to realize the high-throughput one-to-one pairing of single cells and single microspheres in microwell plates. Fluorescence-activated flow cytometry, which is widely used for microparticle sorting, is not suitable for the sorting of encoded microspheres. First, the cost of encoding microspheres is expensive. Since fluorescence-activated flow sorting requires a large amount of background microspheres, this method will cause a lot of waste of reagents. Second, the sorting efficiency is low. Secondly, the single microspheres sorted are easily broken. Unable to achieve high-efficiency high-throughput single cell and single microsphere rapid one-to-one pairing

However, the limitations of conventional microfluidic chips are also very obvious. It needs to design pumps and valves inside the chip to cooperate with external fluid control equipment with complex operations. The technical threshold is high, and it is difficult to reuse a chip and consume a lot of Chip fabrication costs (Macosko et al., 2015, Cell, 161, 1202-1214; Klein et al., 2015, Cell, 161, 1187-1201; Han et al., 2018, Cell, 172, 1091–1107)

[0008] The fourth potential technical problem is that when the previous sequencing methods were used to analyze actual samples, the currently reported sequencing methods based on encoded microspheres all need to use fluorescence-activated flow cytometry to sort the target cells first, and then transfer them to each cell. On the analysis platform, the information of the cell contents will change with the change of the environment of the cell, resulting in the sequencing information reflected in the final sequencing result may be biased compared with the information in the real environment of the cell at that time

[0009] The fifth technical difficulty lies in the separation of rare cells. When the number of cells to be analyzed is very rare and the independent transcriptome information of each single cell needs to be analyzed, traditional techniques based on capillary picking, gradient dilution, or laser cutting require labor costs. High, time-consuming and labor-intensive, low throughput, which limits the high-throughput rapid separation and sequencing analysis of rare cells

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